Discontinuation of Smoking Increases the Risk for Developing Thyroid Peroxidase Antibodies and/or Thyroglobulin Antibodies: A Prospective Study

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1 ORIGINAL ARTICLE Endocrine Care Brief Report Discontinuation of Smoking Increases the Risk for Developing Thyroid Peroxidase Antibodies and/or Thyroglobulin Antibodies: A Prospective Study Grigoris Effraimidis, Jan G. P. Tijssen, and Wilmar M. Wiersinga Departments of Endocrinology and Metabolism (G.E., W.M.W.), and Cardiology (J.G.P.T.), Academical Medical Center, University of Amsterdam, 1105 AZ Amsterdam Zuidoost, The Netherlands Context: Autoimmune thyroid disease develops in genetic susceptible subjects, provoked by environmental factors. Little is known of the environment in the early stages of autoimmunity. Objective: We evaluated environmental factors contributing to de novo occurrence of thyroid antibodies. Design: We conducted a prospective cohort study of 521 euthyroid women without thyroid antibodies in serum who were relatives of autoimmune thyroid disease patients. Follow-up was 5 yr. Baseline characteristics were related to the occurrence of thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies. Exposure to environmental factors in the year prior to the occurrence of antibodies was investigated in a nested case-control study. Results: The 5-yr probability for conversion to TPO antibodies (TPO-Ab) and/or Tg antibodies (Tg-Ab) was 20.1%, and for TPO-Ab alone the probability was 14.5%. None of the baseline characteristics except TSH contributed to the risk of seroconversion. Each case (occurrence of antibodies) was matched for age and duration of follow-up with two controls (no seroconversion). Exposure to environmental stimuli was similar between cases and controls except for smoking. At study entrance, current smokers among cases and controls were 31.3 and 35.5%, respectively (nonsignificant). Current smoking decreased in cases during follow-up. Consequently, the odds ratios (OR) of smoking for developing TPO-Ab and/or Tg-Ab were 0.62 [95% confidence interval (CI), ] 1 yr before seroconversion and 0.59 (95% CI, ) at seroconversion; for conversion to TPO-Ab, these figures are 0.58 (95% CI, ) and 0.54 (95% CI, ), respectively. Conclusion: Discontinuation of smoking is associated with an increased risk for occurrence of TPO-Ab and/or Tg-Ab in serum. The observation is in line with the decreased risk of hypothyroidism in smokers. (J Clin Endocrinol Metab 94: , 2009) Autoimmune thyroid disease (AITD) results from interplay among genetic and environmental factors. The occurrence of thyroid peroxidase antibodies (TPO-Ab) in serum is one of the earliest signs of thyroid autoimmunity that can be detected (1). To get more insight into the early pathogenesis of AITD, we conducted a prospective 5-yr follow-up study on the novel occurrence of thyroid antibodies in euthyroid women at risk for AITD. Subjects and Methods Participants This study was carried out among the 803 subjects from the Amsterdam AITD Cohort (2, 3), which consisted of women between 18 and 65 yr of age in self-proclaimed good health without a history of thyroid disease, who had at least one first- or second-degree relative with documented autoimmune hyper- or hypothyroidism. ISSN Print X ISSN Online Printed in U.S.A. Copyright 2009 by The Endocrine Society doi: /jc Received July 17, Accepted January 7, First Published Online January 13, 2009 Abbreviations: AITD, Autoimmune thyroid disease; CI, confidence interval; FT 4, free T 4 ; NS, nonsignificant; OR, odds ratio; TBII, TSH binding inhibitory Ig; Tg, thyroglobulin; Tg-Ab, Tg antibodies; TPO, thyroid peroxidase; TPO-Ab, TPO antibodies jcem.endojournals.org J Clin Endocrinol Metab. April 2009, 94(4):

2 J Clin Endocrinol Metab, April 2009, 94(4): jcem.endojournals.org 1325 Subjects were followed for 5 yr, or for less time when overt hyper- or hypothyroidism had occurred [defined as TSH 0.4 mu/liter in combination with free T 4 (FT 4 ) 20.1 pmol/liter, or TSH 5.7 mu/liter in combination with FT pmol/liter, respectively]. Blood samples were collected annually to measure TSH, FT 4,T 3, TPO-Ab, Tg-Ab, and TSH binding inhibitory Ig (TBII), and subjects were asked to fill in questionnaires on smoking habits, use of estrogens, pregnancies, iodine excess, and stress. Participants were selected as follows. We excluded subjects who at study entrance had overt or subclinical hyper- or hypothyroidism, or thyroid antibodies (i.e. serum concentrations of TPO-Ab 100 ku/liter, Tg-Ab 100 ku/liter, or TBII 12 U/liter) and those who had no followup; 521 participants were finally enrolled. We performed two different analyses. The first was a time-to-event analysis (event was defined as the occurrence of serum TPO-Ab and/or Tg-Ab 100 ku/liter); the probability of an event was estimated from characteristics of the 521 participants at study entrance. The second analysis was based on a nested case-control study design. A subject was recruited as a case when she had developed TPO-Ab and/or Tg-Ab. The end-point for a case was the time at which she had become positive for the first time for one of the two antibodies (TPO-Ab or Tg-Ab). Cases and controls (two controls for each case) were matched by age and duration of follow-up (4). Age was calculated as the difference between date of study entry and date of birth in years. Controls should have remained seronegative for thyroid antibodies up to the time when the cases to which they were matched had received their end-point. Subjects who during follow-up developed thyroid antibodies and showed up as cases later, or who developed subclinical or overt thyroid dysfunction, were still qualified to act as controls. A subject could only be sampled once as control. Smoking behavior, pregnancy, and exposure to exogenous estrogens, iodine excess, and stress in the year before the seroconversion were compared between cases and controls. Current smoking was defined as smoking now or having stopped smoking in the year before the study visit. Laboratory measurements Serum TSH and FT 4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland), and total T 3 was measured by an in-house RIA. Reference values are: TSH, mu/liter; FT 4, pmol/liter; and T 3, nmol/liter. TPO-Ab and Tg-Ab were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and anti-tg, respectively; Brahms, Berlin, Germany). Improved versions of both assays became available during follow-up. Detection limits of these new assays were: TPO-Ab, 30 ku/liter; and Tg-Ab, 20 ku/liter. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values above 100 ku/liter. TSH receptor antibodies were determined as TBII using the TRAK assay (Brahms); detection limits in the first and second generation TRAK assays were 5 and 1 IU/liter, respectively, and values above 12 and 1.5 U/liter, respectively, were considered as positive. Stress assessments have been published in detail previously (5). Statistical analysis Data processing was done with the statistical software SPSS for Windows, version 12.0 (SPSS, Inc., Chicago, IL). Normally distributed data are presented as mean SD and were analyzed by Student s t test. Data that are not distributed normally are expressed as median and 25th and 75th percentiles and were analyzed by Mann-Whitney U test. Categorical data are expressed as percentages. The significance of differences between groups was analyzed with the 2 test or with Fisher exact test in the case of small numbers. Prognostic indicators for seroconversion to positive thyroid Abs were identified using Cox regression analysis, univariate and multivariate if necessary. Statistical significance was set at 5%. Results Cohort study At study entrance, mean age was yr, and mean serum TSH was mu/liter. During follow-up, 17 subjects (3.3%) developed both TPO-Ab and Tg-Ab, 53 subjects (10.2%) developed only TPO-Ab, and 29 (5.6%) developed only Tg-Ab. At the time of seroconversion, the median TPO-Ab value was 125 ku/liter (range, ku/liter), and the median Tg-Ab value was ku/liter (range, ku/liter). Kaplan-Meier analysis indicated a 5-yr probability of seroconversion of 20.1% for TPO-Ab and/or Tg-Ab, and 14.5% for TPO-Ab only. Prognostic indicators at study entrance for conversion during follow-up to TPO-Ab and/or Tg-Ab and to TPO-Ab independent of Tg-Ab status were identified by Cox regression analysis. Age did not contribute to the risk of seroconversion, nor did any of the environmental factors (smoking, estrogen medication, pregnancy, iodine excess, stress) affect the hazard ratio significantly (Table 1, data on stress not shown). Only baseline serum TSH was positively related to the risk of seroconversion by univariate analysis. Nested case-control study There was a perfect match with respect to age and duration of follow-up between cases and controls (Table 2). The median time to seroconversion in cases was 2.20 yr (for developing either TPO-Ab or Tg-Ab) and 2.98 yr (for developing TPO-Ab independent of Tg-Ab). Serum TSH increased during follow-up in cases but not in controls; serum FT 4 did not change in the follow-up in either group. At study entrance, the frequency of smokers among cases was comparable to that in controls [31.3 vs. 35.5%, nonsignificant (NS)]. At 1 yr before seroconversion, there were less current smokers in cases than in controls (28.3 vs. 39.0%; P 0.068), and this was still true at the time of seroconversion (27.3 vs. 39.0%; P 0.046). The OR of smoking for developing TPO-Ab and/or Tg-Ab were 0.62 [95% confidence interval (CI), ] 1 yr before the seroconversion, and 0.59 (95% CI, ) at seroconversion. When only conversion to TPO-Ab is considered, the OR of smoking were 0.58 (95% CI, ) 1 yr before seroconversion and 0.54 (95% CI, ) at the time of seroconversion. The protective effects of smoking on seroconversion were maintained in a multivariate logistic regression analysis adjusting for pregnancy and estrogen use (data not shown). Discussion The main finding of the present study was that discontinuation of smoking increases the risk of de novo occurrence of serum TPO-Ab and/or Tg-Ab in subjects susceptible for developing AITD. At study entrance, smoking behavior did not differ between those who subsequently developed thyroid antibodies and those who did not; the effect of smoking became obvious only during follow-up. A limitation of our study is that we have not looked after a dose-response effect between smoking and the occurrence of thyroid antibodies; the relatively small number of subjects who changed their smoking behavior during the 5-yr follow-up did not allow a meaningful analysis. The still modest

3 1326 Effraimidis et al. Smoking and the Risk of Developing TPO-Ab and Tg-Ab J Clin Endocrinol Metab, April 2009, 94(4): TABLE 1. Baseline characteristics, event rates, and Cox regression analysis in a prospective cohort of 521 euthyroid women with first- or second-degree relatives with proven AITD who were followed for 5 yr TPO-Ab and/or Tg-Ab TPO-Ab n Event rate (%) HR 95% CI P value Event rate (%) HR 95% CI P value All women Age (yr) 30 a b 11.8 b b b b b b b b b TSH (mu/liter) a c 12.1 d c d c d c d Smoking habits Never smoker a Ex-smoker Current smoker Estrogen medication Never use a Ex use Current use Gravida Nullipara a Uni- and multipara Iodine excess Absent a Present HR, Hazard ratio. a This served as the reference group (category). b P value for trend: NS. c P value for trend: d P value for trend: sample size of the case-control study on conversion to TPO-Ab may also explain why the upper value of the 95% CI of the OR did not decrease below the value of 1.00 but remained 1.02 (it decreased 1.00 when conversion to TPO-Ab and/or Tg-Ab was studied). In view of the significant fall in OR during follow-up in both studies, we feel however quite confident about the validity of our conclusion. Another finding of the present study is that at study entrance a serum TSH level above 2 mu/liter but still within the normal reference range already indicates a small risk for developing thyroid antibodies in the next 5 yr. Thyroid ultrasound may detect signs of thyroid autoimmunity before thyroid antibodies appear in serum (6). We did not perform thyroid ultrasonography, which is another restriction to our study findings. These limitations are in our view well balanced by the strengths of our study. Its prospective nature guarantees more solid evidence than obtained from cross-sectional studies. Moreover, in the nested case-control study a perfect match existed between both groups with respect to age and duration of followup. Higher age and longer exposure time both increase the likelihood of developing thyroid antibodies, constituting possible bias. The matching procedure effectively excluded both biases. The present study is the first to evaluate the relationship between smoking and thyroid antibodies in a prospective manner, but three cross-sectional studies on this topic have been published with essentially similar conclusions. A decreased prevalence of TPO-Ab in smokers compared with nonsmokers was reported among women of the Amsterdam AITD Cohort (2). The OR of current smoking for the presence of TPO-Ab was 0.69 (95% CI, ). Data from the third National Health and Nutrition Examination Survey (NHANES III) (7) indicated that fewer smokers (11%; 95% CI, 10 13) had TPO-Ab and/or Tg-Ab compared with nonsmokers (18%; 95% CI, 17 19). The relationship persisted when analyzing the presence of one antibody independently of the status of the other antibody. Recently, a Danish population study (8) found that smoking was negatively associated with the presence of thyroid antibodies, with the strongest association between smoking and Tg-Ab (OR, ). Taken together, the various studies provide good evidence that smoking decreases the prevalence (crosssectional studies) and incidence (our present study) of TPO-Ab and Tg-Ab. One may thus ask the question whether smoking decreases the risk of hypothyroidism. A meta-analysis could not prove such an association (9). A Danish study found a lower prevalence of mild hypothyroidism (TSH 3.6 mu/liter) among smokers compared with nonsmokers (2.6 vs. 5.3%) with an adjusted OR of 0.47 (95%

4 J Clin Endocrinol Metab, April 2009, 94(4): jcem.endojournals.org 1327 TABLE 2. Comparison of characteristics between cases (seroconverters) and controls (nonseroconverters matched for age and duration of follow-up) at baseline, 1 yr before event (seroconversion), and at the time of event Baseline 1 yr before event At event P value Cases Controls Cases Controls Cases Controls Baseline 1 yr before event At event TPO-Ab and/or Tg-Ab Age (yr) TSH (geometric mean) 1.22 ( ) 1.19 ( ) 1.25 ( ) 1.16 ( ) 1.28 ( ) 1.15 ( ) FT 4 (pmol/liter) Current smokers (%) Pregnant last year (%) Estrogen use (%) Iodine excess (%) Positive and negative affect schedule scale (tendency to report) Negative feelings 20.0 ( ) 21.0 ( ) 21.0 ( ) 21.0 ( ) 20.0 ( ) 20.0 ( ) Positive feelings 39.0 ( ) 38.0 ( ) 39.0 ( ) 38.0 ( ) 37.0 ( ) 37.0 ( ) No. of recent life events unpleasantness pleasantness Daily hassles, total intensity 10.0 ( ) 10.0 ( ) 9.0 ( ) 9.0 ( ) 8.0 ( ) 8.0 ( ) ( ) 15.0 ( ) 12.1 ( ) 13.0 ( ) 12.5 ( ) 11.0 ( ) ( ) 16.0 ( ) 14.0 ( ) 14.0 ( ) 13.0 ( ) 14.0 ( ) ( ) 27.0 ( ) 31.5 ( ) 28.0 ( ) 32.0 ( ) 30.0 ( ) TPO-Ab Age (yr) TSH (geometric mean) 1.21 ( ) 1.21 ( ) 1.22 ( ) 1.17 ( ) 1.28 ( ) 1.15 ( ) FT 4 (pmol/liter) Current smokers (%) Pregnant last year (%) Estrogen use (%) Iodine excess (%) Positive and negative affect schedule scale (tendency to report) Negative feelings 21.0 ( ) 21.0 ( ) 21.0 ( ) 20.0 ( ) 19.0 ( ) 21.0 ( ) Positive feelings 39.0 ( ) 39.0 ( ) 39.0 ( ) 38.0 ( ) 37.0 ( ) 37.0 ( ) No. of recent life events unpleasantness pleasantness Daily hassles, total intensity 10.0 ( ) 11.0 ( ) 9.5 ( ) 9.0 ( ) 8.0 ( ) 8.0 ( ) ( ) 16.0 ( ) 12.0 ( ) 13.0 ( ) 12.0 ( ) 13.0 ( ) ( ) 16.0 ( ) 14.0 ( ) 14.0 ( ) 13.0 ( ) 14.0 ( ) ( ) 27.0 ( ) 31.5 ( ) 29.0 ( ) 32.0 ( ) 31.0 ( ) P values indicate differences between cases and controls at each time point.

5 1328 Effraimidis et al. Smoking and the Risk of Developing TPO-Ab and Tg-Ab J Clin Endocrinol Metab, April 2009, 94(4): CI, ) (10). In NHANES III smokers had less frequently an elevated TSH value ( 4.5 mu/liter) compared with nonsmokers (2.6%; 95% CI, %; vs. 5.5%; 95% CI, ) (7). Recently, a cross-sectional population-based study from Norway reported a lower prevalence of overt and subclinical hypothyroidism among current smokers compared with never smokers (OR, 0.60; 95% CI, for overt; and OR, 0.54; 95% CI, for subclinical hypothyroidism in women; and OR, 0.51; 95% CI, ; and OR, 0.37; 95% CI, , respectively in men) (11). One may therefore hypothesize that the preventive effect of smoking is, at least partly, explained by the protective effect of smoking on the development of TPO-Ab and Tg-Ab. The findings in hypothyroidism are in sharp contrast with many studies showing that smoking is a risk factor for the development of Graves hyperthyroidism, especially for Graves ophthalmopathy (12). Apparently, smoking behavior may determine to a certain extent the clinical phenotype of AITD: smokers are less likely to develop hypothyroidism but more likely to get Graves disease. The mechanism behind these divergent effects of smoking is poorly understood. Acknowledgments Address all correspondence and requests for reprints to: Wilmar M. Wiersinga, Department of Endocrinology, F5-171, Academic Medical Center, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. w.m.wiersinga@amc.uva.nl. Disclosure Summary: The authors have nothing to disclose. References 1. Vanderpump MP, Tunbridge WMG, French JM, Appleton D, Bates D, Clark F, Evans, JG, Hasan DM, Rodgers H, Tunbridge F, Young ET 1995 The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 43: Strieder TG, Prummel MF, Tijssen JG, Endert E, Wiersinga WM 2003 Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol (Oxf) 59: Strieder TG, Tijssen JG, Wenzel BW, Endert E, Wiersinga WM 2008 Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 168: Hollowell JG, Staehling NW, Flanders WD, Hannon WH, Gunter EW, Spencer CA, Braverman LE 2002 Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 87: Strieder TG, Prummel MF, Tijssen JG, Brosschot JF, Wiersinga WM 2005 Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women. Brain Behav Immun 19: Marcocci C, Vitti P, Cetani F, Catalano F, Concetti R, Pinchera A 1991 Thyroid ultrasonography helps to identify patients with diffuse lymphocytic thyroiditis who are prone to develop hypothyroidism. J Clin Endocrinol Metab 72: Belin RM, Astor BC, Powe NR, Ladenson PW 2004 Smoke exposure is associated with a lower prevalence of serum thyroid autoantibodies and thyrotropin concentration elevation and a higher prevalence of mild thyrotropin concentration suppression in the third National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 89: Pedersen IB, Laurberg P, Knudsen N, Jorgensen T, Perrild H, Ovesen L, Rasmussen LB 2008 Smoking is negatively associated with the presence of thyroglobulin autoantibody and to a lesser degree with thyroid peroxidase autoantibody in serum: a population study. Eur J Endocrinol 158: Vestergaard P 2002 Smoking and thyroid disorders a meta-analysis. Eur J Endocrinol 146: Knudsen N, Bulow I, Laurberg P, Perrild H, Ovesen L, Jorgensen T 2002 High occurrence of thyroid multinodularity and low occurrence of subclinical hypothyroidism among tobacco smokers in a large population study. J Endocrinol 175: Asvold BO, Bjoro T, Nilsen TI, Vatten LJ 2007 Tobacco smoking and thyroid function: a population-based study. Arch Intern Med 167: Prummel MF, Wiersinga WM 1993 Smoking and risk of Graves disease. JAMA 269: